CN116773811A

CN116773811A - Application of BRCA2 truncated protein in lung cancer screening

Info

Publication number: CN116773811A
Application number: CN202310388396.0A
Authority: CN
Inventors: 孙苏彭; 康美华; 阴亮; 孙立平
Original assignee: SHANGHAI HENGXIN BIOTECHNOLOGY CO Ltd
Current assignee: SHANGHAI HENGXIN BIOTECHNOLOGY CO Ltd
Priority date: 2023-04-12
Filing date: 2023-04-12
Publication date: 2023-09-19

Abstract

The application relates to application of BRCA2 truncated protein in lung cancer screening. Specifically, the application provides application of an Anti-BRCA2 detection reagent, wherein the detection reagent is BRCA2 truncated protein. The truncated protein has high affinity to Anti-BRCA2, and has high sensitivity and high specificity in lung cancer detection, thereby being beneficial to more accurately and earlier diagnosis of lung cancer.

Description

Application of BRCA2 truncated protein in lung cancer screening

Technical Field

The application relates to the field of in-vitro diagnostic reagents, in particular to application of BRCA2 truncated protein in lung cancer screening.

Background

Lung cancer is one of the most common malignant tumors in the world, the morbidity and mortality of the lung cancer are in an increasing trend year by year, and the morbidity is the first in the world at present, so that the lung cancer seriously threatens the health and life of human beings.

Lung cancer is a disease which is good at hiding, clinical symptoms are often shown when the disease progresses to late stage, 70-80% of lung cancer patients are in middle and late stages when the lung cancer symptoms are diagnosed, cancer cells are spread, the best cure time is missed, and the five-year survival rate is low. For early lung cancer patients, the survival rate and the survival quality of the patients can be greatly improved by 5 years or more after timely treatment. Early diagnosis and effective screening of lung cancer is therefore of paramount importance.

Thus, there is a great need in the art to develop new biomarkers, detection reagents and detection techniques that can be used for early diagnosis or screening of lung cancer.

Disclosure of Invention

The application aims to provide novel biomarkers, detection reagents and detection technologies for early diagnosis or screening of lung cancer.

In a first aspect of the application, there is provided the use of an Anti-BRCA2 detection reagent for the preparation of a detection reagent/kit for detecting lung cancer; wherein, the detection reagent is BRCA2 truncated protein.

In another preferred embodiment, the truncated protein is a non-full length protein.

In another preferred embodiment, the ratio of the length L1 of the truncated BRCA2 protein to the length L0 of the full-length BRCA2 protein is less than or equal to 1/2, preferably less than or equal to 1/3, and more preferably less than or equal to 1/4.

In another preferred embodiment, the detection reagent comprises one or more BRCA2 truncated proteins.

In another preferred embodiment, the BRCA2 truncated protein comprises the following fragment of SEQ ID No. 2: bits 1-637, 1500-1908, 1700-1741, 1720-1770, 3160-3418, 1700-1800, 1700-1838, 2279-3418, or 2840-3418.

In another preferred embodiment, the BRCA2 truncated protein comprises the following fragment of SEQ ID No. 2: 1720-1770, 3160-3418, 1700-1800, or 1700-1838.

In another preferred embodiment, the BRCA2 truncated protein comprises a fragment from position Z1 to position ZN in SEQ ID No. 2, wherein Z1 is any positive integer from 1700 to 1720 and ZN is any positive integer from 1770 to 1838.

In another preferred embodiment, the BRCA2 truncated protein is selected from the group consisting of:

(Y1) truncated protein 6, the amino acid sequence of which is amino acid 1720-1770 of BRCA2 protein;

(Y2) truncated protein 9, the amino acid sequence of which is amino acids 3160-3418 of BRCA2 protein;

(Y3) truncated protein 3, the amino acid sequence of which is amino acids 1700-1800 of BRCA2 protein;

(Y4) truncated protein 4, the amino acid sequence of which is amino acids 1700-1838 of BRCA2 protein;

(Y5) any combination of the above Y1 to Y4;

wherein the amino acid sequence of the BRCA2 protein is shown as SEQ ID No. 2.

In another preferred embodiment, and the truncated protein comprises:

(Y2) truncated protein 9, the amino acid sequence of which is amino acids 3160-3418 of BRCA2 protein; and

(Ya) any one of the truncated proteins selected from the group consisting of:

(Y4) truncated protein 4, the amino acid sequence of which is amino acids 1700-1838 of BRCA2 protein.

In another preferred embodiment, the BRCA2 truncated protein comprises: (Y2) truncated protein 9, the amino acid sequence of which is amino acids 3160-3418 of BRCA2 protein; and (Y1) truncated protein 6, the amino acid sequence of which is amino acid 1720-1770 of BRCA2 protein.

In another preferred embodiment, the detection of lung cancer is the detection of the level of Anti-BRCA2 in the test sample.

In another preferred embodiment, the sample to be tested includes: blood samples, plasma samples and serum samples.

In another preferred embodiment, the lung cancer patient is an Anti-BRCA2 antibody positive patient.

In another preferred embodiment, the lung cancer patient is a male patient or a female patient.

In another preferred embodiment, the assay kit contains a kit of parts, which describes the assay method.

In a second aspect of the application, there is provided an isolated human BRCA2 truncated protein selected from the group consisting of:

(A1) A polypeptide having amino acids 1720 to 1770 of the amino acid sequence of SEQ ID NO. 2;

(A2) A polypeptide having amino acids 3160 to 3418 of the amino acid sequence of SEQ ID NO. 2;

(A3) A polypeptide having amino acids 1700-1800 of the amino acid sequence of SEQ ID NO. 2;

(A4) A polypeptide having amino acids 1700-1838 of the amino acid sequence of SEQ ID NO. 2.

In another preferred embodiment, the truncated protein is a polypeptide having amino acids 1720 to 1770 of the amino acid sequence of SEQ ID NO. 1.

In a third aspect of the application, there is provided an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of:

(a) A polynucleotide encoding a truncated protein according to the second aspect of the application;

(b) A polynucleotide complementary to polynucleotide (a).

In another preferred embodiment, the polynucleotide encodes a polypeptide selected from the group consisting of:

In another preferred embodiment, the polynucleotide has the sequence corresponding to the coding sequence of SEQ ID No. 1.

In a fourth aspect of the application there is provided a vector comprising a polynucleotide according to the third aspect of the application.

In a fifth aspect of the application there is provided a genetically engineered host cell comprising a vector according to the fourth aspect of the application or having integrated into its genome a polynucleotide according to the third aspect of the application.

In another preferred embodiment, the host cell is a prokaryotic cell or a eukaryotic cell.

In another preferred embodiment, the host cell is E.coli or yeast.

In a sixth aspect of the present application, there is provided a method for preparing BRCA2 truncated protein, the method comprising:

(a) Culturing the host cell of the fifth aspect of the application under conditions suitable for expression;

(b) Isolating said BRCA2 truncated protein from the culture.

In a seventh aspect of the application, there is provided a kit comprising:

(a) The kit comprises a first container and an Anti-BRCA2 detection reagent in the first container, wherein the detection reagent is BRCA2 truncated protein;

(b) A description of the detection method is given.

In another preferred embodiment, the detection method is to detect the level of Anti-BRCA2 antibodies in the sample to be tested and is used for early screening or assisted diagnosis of lung cancer.

(Y1) truncated protein 6, the amino acid sequence of which is amino acid 1720-1770 of BRCA2 protein; and/or

(Y2) truncated protein 9, the amino acid sequence of which is 3160 th to 3418 th amino acids of BRCA2 protein.

In an eighth aspect of the present application, there is provided a detection reagent for lung cancer detection,

the detection reagent is BRCA2 truncated protein, and the BRCA2 truncated protein comprises:

(Ya) any one of the truncated proteins selected from the group consisting of:

In a ninth aspect of the application, there is provided a kit according to the seventh aspect of the application or a use of a detection reagent according to the eighth aspect of the application, for the preparation of a kit for early screening or assisted diagnosis of lung cancer by detecting the level of Anti-BRCA2 in a sample to be tested.

It is understood that within the scope of the present application, the above-described technical features of the present application and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows the truncation sites of BRCA2 autoantibodies.

FIG. 2 shows a scatter plot obtained after the reaction of the truncated proteins of various BRCA2 of the present application with serum from healthy, benign, or lung cancer populations. FIG. 2A shows a scatter plot of BRCA2 truncated proteins 1-637, 1500-1908, 1700-1800, and 1700-1838; FIG. 2B shows a scatter plot of BRCA2 truncated proteins 1700-1741, 1720-1770, 2279-3418, 2840-3418, and 3160-3418.

FIG. 3 shows the working curves (receiver operating characteristic curve, abbreviated ROC curves) of subjects with four BRCA2 fragments (1700-1800, 1700-1838, 1720-1770 and 3160-3418) that are significantly elevated and have a strong epitope activity in lung cancer serum.

Detailed Description

The present inventors have conducted extensive and intensive studies and, as a result of extensive screening, have unexpectedly developed a BRCA2 truncated protein capable of binding highly specifically to an autoantibody against BRCA2 (Anti-BRCA 2) of a lung cancer patient. The truncated protein of the application has shorter amino acid sequence and smaller molecular weight, can have high specific affinity with Anti-BRCA2 antibodies in serum, and has no or basically no cross reaction with other antibodies in blood or serum, thus being capable of remarkably improving the sensitivity and specificity of diagnosis and screening of lung cancer. The present application has been completed on the basis of this finding.

Experiments show that various BRCA2 truncated proteins (such as BRCA2 truncated proteins 1700-1800, 1700-1838, 1720-1770 and 3160-3418) can be used for detecting Anti-BRCA2 in a serum sample more effectively and sensitively in early stage, so that early stage screening and auxiliary diagnosis of lung cancer can be realized.

Terminology

As used herein, the terms "comprising," "including," and "includes" can be open, closed, or semi-closed. The term may thus also include "consisting of …" or "consisting essentially of …".

Autoantibodies

Autoantibodies refer to antibodies produced by the body against an organ, cell or cellular component of the body.

Currently, autoantibodies to certain proteins have become markers for lung cancer, such as autoantibodies to the following proteins: p53, NY-ESO-1, CYFRA, etc.

As used herein, the terms "Anti-BRCA2", "autoantibody to BRCA2" are used interchangeably to refer to the production of an autoantibody to BRCA2 in a subject (e.g., a human). It will be appreciated that Anti-BRCA2 generally includes a variety of different Anti-BRCA2, which different Anti-BRCA2 will tend to be directed to different binding sites.

Studies of the present application have shown that in the blood of certain lung cancer patients, autoantibodies against BRCA2 (Anti-BRCA 2 antibodies) are present, which can serve as biomarkers for the detection of lung cancer. Further, the inventors have unexpectedly found that certain truncated proteins of BRCA2 can bind to these Anti-BRCA2 in blood samples with high specificity and high affinity, and are therefore particularly suitable for use in early screening and assisted diagnosis of lung cancer.

Human breast cancer susceptibility gene 2 (BRCA 2) and protein

As used herein, the terms "BRCA2 protein", "BRCA2 polypeptide" are used interchangeably to refer to a protein or polypeptide having the amino acid sequence (SEQ ID NO: 2) of human breast cancer susceptible protein BRCA2.

As used herein, the term "BRCA2 truncated protein", or "BRCA2 truncated polypeptide", "BRCA2 truncate", or "BRCA2 truncate", "BRCA2 truncated mutant (truncated variant)" is used interchangeably to refer to a polypeptide formed by truncating the amino acid sequence of the breast cancer susceptible protein BRCA2.

Human breast cancer susceptibility gene 2 (BRCA 2), a susceptibility gene for genetically predisposed breast and ovarian cancers, in a mutant form that may lead to an increased risk of developing these cancers. The BRCA2 gene is located at 13 th chromosome 13q12.3, has a total length of 84,193bp, has an mRNA length of 11,3836 nt, and has 27 exons, and encodes a protein composed of 3418 amino acid residues (SEQ ID NO: P51587 in UniProtKB database).

The nucleotide sequence and the amino acid sequence of human BRCA2 are respectively shown in SEQ ID NO:1 and SEQ ID NO: 2.

Truncated proteins of the application

As used herein, "truncated protein of the application," "truncated polypeptide of the application," and "polypeptide of the application" are used interchangeably, and are polypeptides formed by truncating the amino acid sequence of BRCA2, which can react with Anti-BRCA2 in the blood of a patient with lung cancer, and thus be used to qualitatively or quantitatively detect Anti-BRCA2 in a sample to be tested.

As used herein, "truncated proteins of the application" excludes native proteins. And is also provided with

As used herein, the term "BRCA2 truncated protein 1700-1800" refers to a truncated protein formed from the amino acid sequence at positions 1700-1800 after cleavage of the amino acid sequence of the BRCA2 protein. The terms "BRCA2 truncated proteins 1700-1838", "BRCA2 truncated proteins 1720-1770" and "BRCA2 truncated proteins 3160-3418" are similar.

In the present application, a suitable sample to be tested is a blood sample or any sample derived from blood and containing an antibody component in the blood. Representative samples to be tested include (but are not limited to): blood samples, plasma samples and serum samples.

As used herein, "isolated" refers to a substance that is separated from its original environment (i.e., the natural environment if it is a natural substance). If the naturally occurring polynucleotide and polypeptide are not isolated or purified in vivo, the same polynucleotide or polypeptide is isolated or purified from other naturally occurring substances.

As used herein, "isolated polypeptide of the application" means that the BRCA2 truncated protein of the application is substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. Those skilled in the art can purify the polypeptides of the application using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel.

The polypeptide of the present application may be a recombinant polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. The polypeptides of the application may be chemically synthesized products (e.g., prepared using peptide synthesis techniques), or produced from prokaryotic or eukaryotic hosts (e.g., bacterial, yeast, higher plant, insect, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptides of the application may be glycosylated or may be non-glycosylated. The polypeptides of the application may or may not also include an initial methionine residue.

The application also includes fragments, derivatives and analogues of the BRCA2 truncated proteins of the application. As used herein, the terms "fragment," "derivative" and "analog" refer to polypeptides that retain substantially the same activity of binding to Anti-BRCA2 as the BRCA2 truncated proteins of the application. The polypeptide fragments, derivatives or analogues of the application may be (i) polypeptides having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) polypeptides having a substituent in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound, such as a compound that extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein.

In the present application, "BRCA2 truncated protein" also includes a variant having the same function as the BRCA2 truncated protein of the present application as Anti-BRCA2 binding. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 30, preferably 1 to 25, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal end. For example, substitution with a small number of amino acids of similar or similar properties will not normally alter the binding function of the BRCA2 truncated proteins of the application to Anti-BRCA2. As another example, the addition of one or more amino acids at the C-terminus and/or N-terminus will not normally alter the binding activity of the BRCA2 truncated proteins of the application to Anti-BRCA2.

The application also provides analogs of BRCA2 truncated proteins. These analogs may differ from the BRCA2 truncated proteins of the application by differences in amino acid sequence, by differences in modified forms that do not affect the sequence, or by both. Analogs also include analogs having residues other than the natural L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the polypeptides of the present application are not limited to the representative polypeptides exemplified above.

Modified (typically without altering the primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during synthesis and processing of the polypeptide or during further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (e.g., mammalian glycosylase or deglycosylase).

In the present application, a "conservative variant polypeptide of a BRCA2 truncated protein" refers to a polypeptide in which up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced with amino acids of similar or similar nature, as compared to the amino acid sequence of the corresponding BRCA2 truncated protein (exemplified by the sequence at positions 3160-3418 or 1720-1770 of SEQ ID NO:2, with other BRCA2 truncated proteins being similar). These conservatively mutated polypeptides are preferably produced by amino acid substitution according to Table 1.

TABLE 1

The polynucleotides of the application may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. As used herein, a "degenerate variant" refers to a nucleic acid sequence that encodes a protein having a truncated BRCA2, but differs from the sequence of the coding region of wild-type human BRCA2.

The term "polynucleotide encoding a polypeptide" may include polynucleotides encoding the polypeptide, or may include additional coding and/or non-coding sequences.

The application also relates to nucleic acid fragments which hybridize to the sequences described above. As used herein, a "nucleic acid fragment" is at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more in length. The nucleic acid fragments may be used in nucleic acid amplification techniques (e.g., PCR) to obtain and/or isolate polynucleotides encoding the BRCA2 truncated proteins of the application.

The polypeptides and polynucleotides of the application are preferably provided in isolated form, and more preferably purified to homogeneity.

The nucleotide sequence of the BRCA2 truncated protein of the application can be obtained usually by PCR amplification, recombinant or artificial synthesis. For the PCR amplification method, primers can be designed according to the nucleotide sequences disclosed in the present application, particularly the open reading frame sequences, and amplified to obtain the relevant sequences using a commercially available cDNA library or cDNA samples prepared according to conventional methods known to those skilled in the art as templates.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

At present, it is already possible to obtain the DNA sequences encoding the proteins of the application (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the application by chemical synthesis.

Methods of amplifying DNA/RNA using PCR techniques are preferred for obtaining the genes of the present application. Primers for PCR can be appropriately selected according to the sequence information of the present application disclosed herein, and can be synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.

The application also relates to vectors comprising the polynucleotides of the application, as well as host cells genetically engineered with the vectors of the application or BRCA2 truncated protein coding sequences, and methods for producing the polypeptides of the application by recombinant techniques.

The polynucleotide sequences of the present application may be used to express or produce recombinant BRCA2 truncated proteins by conventional recombinant DNA techniques. Generally, there are the following steps:

(1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a human BRCA2 truncated protein of the application, or with a recombinant expression vector comprising the polynucleotide;

(2) Host cells cultured in a suitable medium;

(3) Isolating and purifying the protein from the culture medium or the cells.

In the present application, the polynucleotide sequence encoding the BRCA2 truncated protein may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. Suitable vectors for use in the present application include, but are not limited to: t7-based expression vectors expressed in bacteria (Rosenberg, et al Gene,1987, 56:125); pMSXND expression vectors expressed in mammalian cells (Lee and Nathans, J Bio chem.263:3521,1988) and baculovirus-derived vectors expressed in insect cells. In general, any plasmid or vector can be used as long as it replicates and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translational control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequence for BRCA2 truncated proteins and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like (Sambrook, et al molecular Cloning, aLaboratory Manual, cold Spring Harbor laboratory New York, 1989). The DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.coli; a lambda phage PL promoter; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, LTRs from retroviruses, and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or viruses thereof. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences as described above, as well as appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; a plant cell; insect cells of Drosophila S2 or Sf 9; CHO, COS, 293 cells, or Bowes melanoma cells.

When the polynucleotide of the present application is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to increase the transcription of a gene. Examples include the SV40 enhancer 100 to 270 base pairs on the late side of the origin of replication, the polyoma enhancer on the late side of the origin of replication, and adenovirus enhancers.

It will be clear to a person of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli,competent cells capable of DNA uptake can be harvested after exponential growth phase and can be obtained with CaCl ₂ The process is carried out using procedures well known in the art. Another approach is to use MgCl ₂ . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present application. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.

Diagnostic applications

The BRCA2 truncated protein or polypeptide has multiple uses. Such uses include (but are not limited to): the presence or absence of Anti-BRCA2 in the blood sample is detected, thereby providing useful information for early diagnosis or aiding in the diagnosis of lung cancer.

The application also relates to diagnostic assays for quantitative or qualitative detection of Anti-BRCA2 levels. Such tests are well known in the art.

One method of detecting the presence or absence of Anti-BRCA2 in a sample is to use specific binding of the BRCA2 truncated protein to Anti-BRCA2. Typically, the method comprises: contacting a sample (e.g., a plasma or serum sample) with a BRCA2 truncated protein; whether an antibody complex is formed is observed, and the formation of the antibody complex indicates the presence of Anti-BRCA2 in the sample.

Detection agent and kit for diagnosis

The application also provides a detection agent and a kit for early or auxiliary diagnosis of lung cancer, which comprise one or more BRCA2 truncated proteins of the application as detection agents.

The main advantages of the application include:

(a) The truncated protein has high affinity with Anti-BRCA2 in a blood sample of a lung cancer patient.

(b) The truncated protein of the application is almost not combined with other irrelevant antibodies of a blood sample, can reduce nonspecific cross reaction, and improves the signal-to-noise ratio, specificity and accuracy of detection.

(c) The truncated protein can detect Anti-BRCA2 in a blood sample with high sensitivity. (d) The truncated protein of the present application has a short sequence and can be prepared in a large scale by recombinant or polypeptide synthesis.

(e) The diagnosis or detection of lung cancer by using the truncated protein disclosed by the application is carried out only by using a serum or plasma sample, and the detection by taking a tissue sample is not needed, so that the resistance of a patient to the detection is avoided, the early lung cancer patient can be found, the survival period of the patient is prolonged, the life quality is improved, and the truncated protein has a very high clinical application prospect.

(f) The truncated protein of the BRCA2 protein can trigger more obvious antigen-antibody reaction than the BRCA2 protein in the serum of a patient suffering from lung cancer, and particularly the truncated protein positioned in the middle part and the carboxyl end of the whole length of the BRCA2 molecule has higher screening capacity on the lung cancer serum.

The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

Example 1

Design of truncated protein of BRCA2

In this example, based on the sequence of human BRCA2 protein, the present application designed a series of BRCA2 candidate truncated proteins, the candidate truncated sites being as shown in fig. 1.

The nucleic acid sequence of human BRCA2 is shown as SEQ ID NO:1, the amino acid sequence is shown as SEQ ID NO: 2. Some representative secondary structures are shown in table 1.

Table 1 secondary structure

Location (based on SEQ ID No: 2)	Two-stage structure
		31-35	Helix
203-206	Beta strand
		1228-1230	Beta strand
1231-1241	Helix
		1520-1522	Helix
1536-1541	Helix
		1542-1546	Turn
2300-2302	Beta strand
		2311-2314	Helix
2315-2317	Beta strand

In this example, candidate truncated proteins for each BRCA2 are represented by the number of amino acids that they start and terminate, as shown in table 2.

TABLE 2 BRCA2 truncated protein

Example 2

Preparation method of BRCA2 truncated antigen protein

In this example, each BRCA2 truncated protein was prepared by a recombinant method based on the structure of each truncated antigen protein in example 1. The preparation method comprises the following steps:

(1) Construction of recombinant plasmid expressing BRCA2 truncated protein

The nucleotide sequences of the BRCA2 truncated proteins of the lung cancer antigen were prepared by PCR, and the coding sequences of a series of BRCA2 truncated proteins correspond to the 9 truncated proteins of BRCA2 truncated protein 1 to BRCA2 truncated protein 9 shown in table 2.

Extracting plasmid vector containing 6XHIS marked PET28 (a), double-enzyme cutting with Ncol and Ndel, and recovering double-enzyme cut plasmid fragments after electrophoresis; double-enzyme cutting of the BRCA2 truncated protein gene fragment by using Ncol and Ndel, electrophoresis recovery of the double-enzyme cut gene fragment, and standing at-20 ℃ for standby; the double digested plasmid fragment and the double digested gene fragment were ligated overnight at 16℃with T4 ligase at a ratio of 1:3-10, and the recombinant plasmids after ligation were PET28 (a) -BRCA2 (1-637), PET28 (a) -BRCA2 (1500-1908), PET28 (a) -BRCA2 (1700-1800), PET28 (a) -BRCA2 (1700-1838), PET28 (a) -BRCA2 (1700-1741), PET28 (a) -BRCA2 (1720-1770), PET28 (a) -BRCA2 (2279-3418), PET28 (a) -BRCA2 (2840-3418), PET28 (a) -BRCA2 (3160-3418).

(2) Screening and identification of recombinant plasmids

E.coli BL21 (DE 3) was transformed with the recombinant plasmid, and the plasmid was spread on LB plates containing ampicillin (60 ug/ml) and incubated overnight at 37℃in a incubator; the recombinant plasmid transformed colony and the control colony (empty PGEM-5zf plasmid transformed bacteria) are randomly selected the next day, plasmids are respectively extracted, ndeL and Ncol are used for double enzyme digestion, and after enzyme digestion, electrophoresis is performed, so that corresponding target fragments and vectors can be seen, and the construction of the expression vectors is successful, namely the positive expression bacteria.

(3) Efficient expression of recombinant protein engineering bacteria

Inoculating positive expression bacteria into a test tube of 2ml LB culture medium (60 ug/ml ampicillin), oscillating for 4 hours at a constant temperature of 37 ℃ by a shaking table, adding IPTG with a final concentration of 0.5mmol/l, continuously inducing for 6 hours at 37 ℃, centrifugally collecting precipitate bacteria, crushing the precipitate bacteria, taking supernatant and precipitate, respectively carrying out SDS-PAGE detection, and obtaining the expressed target protein in the supernatant.

(4) Purification of expressed proteins

Selecting single colony of recombinant protein engineering bacteria with high expression, inoculating into a triangular flask containing 100ml of LB liquid medium, adding ampicillin to a final concentration of 60ug/ml, and placing in a shaking table at 37 ℃ overnight. Inoculating the bacterial liquid and LB culture medium 1:10 into a triangular flask of 1000ml LB (ampicillin 60 ug/ml) culture liquid for 4 hours, adding IPTG (final concentration is 0.5 mmol/l), continuously inducing for 6 hours at 37 ℃, centrifuging 1000ml engineering bacteria for inducing and expressing fusion protein, centrifuging at a high speed (10000 rpm) for 10 minutes at a low temperature (4 ℃) and suspending the precipitated bacterial liquid in 1/10 lysate (50 mM Tris-HCl, 10mM EDTA, 15mM NaCL and 10mM DTT) of an original centrifugation volume, ice-bathing ultrasonic bacterial bodies, centrifuging at a high speed (12000 rpm) for 30 minutes at a low temperature (4 ℃), and collecting bacterial liquid supernatant.

Washing the balanced nickel ion affinity chromatographic column with a balancing solution (20 mM PB PH 7.4), directly loading the bacterial liquid supernatant collected by ultrasonic treatment on the column at a loading flow rate of 1.5ml/min, washing the column with the balancing solution after loading, eluting proteins with an eluent containing imidazole at concentrations of 20mM, 50mM, 100 mM, 200 mM and 500mM in sequence, collecting the proteins at each eluting peak, and detecting the proteins at each peak by SDS-PAGE electrophoresis.

The results showed that the following recombinant proteins were prepared in high purity: truncated protein 1, truncated protein 2, truncated protein 3, truncated protein 4, truncated protein 5, truncated protein 6, truncated protein 7, truncated protein 8 and truncated protein 9.

Example 3

The truncated proteins 1700-1800, 1700-1838, 1720-1770 and 3160-3418 of BRCA2 have good lung cancer detection specificity

Each recombinant protein purified as prepared in example 2 was diluted with a carbonate buffer (50 mM, PH9.6) and coated on an ELISA plate at a protein level of 100 ul/well and 100 ng/well at 4℃overnight. After washing, 200 ul/well of 10% calf serum PBS was added and blocked at 37℃for 2h, after washing, at 4℃for further use.

The indirect ELISA method for detecting lung cancer comprises 47 patients with benign lung diseases, 25 patients with benign lung diseases (including chronic obstructive pulmonary disease, pulmonary tuberculosis, pneumonia, pneumoconiosis, etc.), and 20 healthy controls matched with the ages of lung cancer cases. Basic information of lung cancer patients is shown in table 3.

TABLE 3 Table 3

	Lung cancer patient (n=47)
		Age range	31～81
Age mean	59
		Sex male	23
Female	24

According to the research of the university of science and technology, same medical college Li Junchuan, et al, BRCA2 has a positive detection rate of 35.7% in a tissue sample of the non-small cell lung cancer, can be used as a detection index of malignant characteristics of the non-small cell lung cancer, and the research result is published in journal of Chinese histochemistry and cytochemistry. According to the team study of Song et al in Jinling Hospital, the mutation rate of BRCA2 gene in non-small cell lung cancer patients is 4.97%, different mutation forms have different effects on prognosis, and both mutation and high expression of BRCA2 can lead the organism to produce autoantibodies, and the result of the study is published in J.pectoral oncology (Journal of Thoracic Oncology).

Indirect ELISA (ELISA) was used to detect the reactivity of BRCA2 autoantibodies in serum of lung cancer patients (47 cases), benign lung disease patients (25 cases including chronic obstructive pulmonary disease, tuberculosis, pneumonia, pneumoconiosis, etc.), and healthy persons (20 cases).

Serum or plasma samples were diluted 1:110 fold with phosphate buffer and reacted by adding microwells (50 ml/well). After washing unbound serum or plasma components with wash solution, horseradish peroxidase (HRP) -conjugated anti-human IgG was added to each well for reaction. Then, TMB (3, 3', 5' -tetramethylbenzidine) as a reaction substrate was added for color development. Stop solution (1N HCl) was added and absorbance was measured at 450nm using a microplate reader (OD).

The results are shown in FIG. 2 and Table 4. After serum responses of healthy, benign and lung cancer groups are respectively carried out on each BRCA2 truncated protein, a scatter diagram shown in figure 2 is obtained, and the truncated proteins generally trigger more obvious antigen-antibody reactions in the serum of a lung cancer patient, namely, OD values are increased in the lung cancer group.

Table 4 (OD value)

	Health care	Benign	Lung cancer	Cutoff
					1-637	0.21	0.25	0.29	0.4
1500-1908	0.29	0.25	0.29	0.6
					1700-1800	0.33	0.26	0.55	1
1700-1838	0.34	0.31	0.67	1
					1700-1741	0.61	0.61	0.73	2.3
1720-1770	0.22	0.36	0.50	0.8
					2279-3418	0.25	0.32	0.26	0.6
2840-3418	0.44	0.42	0.57	1.6
					3160-3418	0.18	0.17	0.26	0.4

Furthermore, the Kruskal-Wallis test shows that the OD signal increase results obtained in serum of lung cancer patients of BRCA2 truncated proteins 1700-1800 (p=0.0356), 1700-1838 (p=0.0270), 1720-1770 (p=0.0012), 3160-3418 (p=0.0417) are statistically significant (P < 0.05). The level of autoantibodies to BRCA2 in the serum of lung cancer patients is significantly higher than in healthy people and patients with benign lung disease.

The results show that the truncated proteins have good reactivity with lung cancer serum, and weak reaction with healthy and benign people, prove that the truncated proteins have good specificity, and can be used for early screening and auxiliary diagnosis of lung cancer.

Example 4

Screening ability evaluation of BRCA2 truncated proteins 1700-1800, 1700-1838, 1720-1770 and 3160-3418 on lung cancer serum

The present inventors analyzed four BRCA2 truncated proteins (or BRCA2 fragments) that were significantly elevated and more active in epitopes in lung cancer serum using subject work profile (ROC): 1700-1800, 1700-1838, 1720-1770 and 3160-3418.

The analysis results are shown in FIG. 3.

The detection performance of each fragment of BRCA2 when the ROC curve was maximal with respect to healthy humans is shown in table 5.

TABLE 5

BRCA2 fragment	Sensitivity (%)	Specificity (%)	Area Under Curve (AUC)
				1700-1800	23.40	95.00	0.6202
1700-1838	31.91	90.00	0.6346
				1720-1770	46.67	90.00	0.7367
3160-3418	27.66	94.74	0.6338

The detection performance of each fragment of BRCA2 when the ROC curve was maximal with respect to benign patients is shown in table 6.

TABLE 6

BRCA2 fragment	Sensitivity (%)	Specificity (%)	Area Under Curve (AUC)
				1700-1800	29.79	92.00	0.6766
1700-1838	34.04	96.00	0.6791
				1720-1770	33.33	100.0	0.7222
3160-3418	25.53	92.00	0.6651

The above results indicate that four preferred BRCA2 truncated proteins (or BRCA2 fragments): 1700-1800, 1700-1838, 1720-1770 and 3160-3418 are useful as detection reagents for early diagnosis or for aiding in the diagnosis of lung cancer for the quantitative or qualitative detection of Anti-BRCA2 in a patient's blood sample.

Furthermore, from the area under each curve in fig. 3, binding sensitivity and specificity analysis revealed: whether a healthy population is used as a control or a benign patient is used as a control, epitopes of BRCA2 molecules located in the middle of the whole length (namely 1700-1838, particularly the core segment of 1720-1770) and at the carboxyl end (namely 3160-3418) can be used for screening lung cancer serum and assisting in diagnosis of lung cancer.

Discussion of the application

The BRCA2 protein is a tumor suppressor, and the protective protein participates in the repair of double-stranded DNA injury by homologous recombination and partial regulation of the activity of Rad51 protein factor, so that the growth and development of cancer cells are prevented.

In lung Cancer studies, mutations in lysine 3326 of BRCA2 were found to correlate with the occurrence of small cell lung Cancer (Rafnar T, sigurjonsdottir GR, stacey SN, et al Association of BRCA K3326. With Small Cell Lung Cancer and Squamous Cell Cancer of the skin. JNAtl Cancer Inst.2018Sep 1;110 (9): 967-974.Doi:10.1093/jnci/djy002.PMID:29767749; PMCID: PMC 6136924.).

Retrospective studies have shown that mRNA and protein expression abnormalities of the BRCA2 gene occur in lung adenocarcinoma, and that promoter hypermethylation is the major mechanism of deregulation of this gene. Abnormal expression of BRCA2 alters the DNA double strand break repair pathway, possibly accompanied by deregulation of interactions with p53 and RB proteins. This mechanism plays an important role in the pathogenesis of part of non-small cell lung Cancer (Lee MN, tseng RC, hsu HS, chen JY, tzao C, ho WL, wang YC. Epigenetic inactivation of the chromosomal stability control genes BRCA, BRCA2, and XRCC5in non-small cell lun Cancer Clin Cancer Res.2007Feb 1;13 (3): 832-8.Doi:10.1158/1078-0432.CCR-05-2694.PMID: 17289874.).

In lung cancer models, the expression level of BRCA2 can be regulated by non-coding RNA, hsa_circ_0046264 can reduce the level of Hsa-miR-1245 through RNA interference phenomenon, so that the increase of BRCA2 protein level can be realized to inhibit tumor growth (Yang L, wang J, fan Y, yu K, jiao B, su X.Hsa_circ_0046264up-regulated BRCA2to suppress lung cancer through targeting Hsa-miR-1245.Respir Res.2018Jun 11;19 (1): 115.doi:10.1186/s12931-018-0819-7.PMID:29891014; PMCID:PMC5996480).

Reports on BRCA2 autoantibodies are mostly found in studies of breast cancer detection, and prior to the application, no related reports on the detection of lung cancer are found, and no prior art related to lung cancer is found.

The present inventors have unexpectedly found that BRCA2 is a lung cancer-associated autoantigen, and that Anti-BRCA2 is present in the blood of lung cancer patients.

Because of the large molecular weight of BRCA2, the yield of prokaryotic cell expression in full length state is low; and a plurality of epitopes are difficult to fully expose in the product of a prokaryotic cell expression system under the full-length state, so that the activity of the protein product for lung cancer screening is lower; however, prokaryotic expression systems are still the most economical and efficient bioreactors at present; secondly, the human immune system will contact a large number of antigen molecules in daily life, and the antigen molecules are inevitably provided with active epitopes similar to lung cancer related antigens, so that the cross reaction of autoantibodies on lung cancer antigens and other antigens can be caused, the specificity of lung cancer screening is reduced, and the lung cancer antigens are subjected to truncated expression to reduce the complexity of the antigen epitopes, so that the probability of cross reaction is greatly reduced.

The inventors have truncated and screened the BRCA2 protein and have unexpectedly found that some specific truncated proteins formed bind with high affinity and high specificity to Anti-BRCA2. The BRCA2 truncated protein has high immunogenicity active epitope, can obviously improve the sensitivity of lung cancer screening, simultaneously reduce nonspecific cross reaction and improve the signal-to-noise ratio, specificity and accuracy of detection.

The truncated protein fragment of the application is easy to be expressed in a recombination way or synthesized by polypeptide, and particularly can obtain high yield in a prokaryotic expression system, thereby being convenient for large-scale production.

Early screening for lung cancer was indicated. The BRCA2 truncated protein can be used for screening lung cancer (especially early screening), is particularly suitable for conventional screening of people without symptoms related to the lung cancer, and can discover the lung cancer as early as possible before symptoms appear, so that a clinician is prompted to take related follow-up diagnosis or corresponding therapeutic measures or decisions on the patient earlier.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims

1. The use of an Anti-BRCA2 detection reagent for preparing a detection reagent/kit for detecting lung cancer; wherein, the detection reagent is BRCA2 truncated protein.

2. The use according to claim 1, wherein the ratio of BRCA2 truncated protein length L1 to BRCA2 full length protein length L0 is +.1/2, preferably +.1/3, more preferably +.1/4.

3. The use according to claim 1, wherein the BRCA2 truncated protein comprises the following fragment of SEQ ID No. 2: 1720-1770, 3160-3418, 1700-1800, or 1700-1838.

4. The use according to claim 1, wherein the BRCA2 truncated protein is selected from the group consisting of:

(Y5) any combination of the above Y1 to Y4;

wherein the amino acid sequence of the BRCA2 protein is shown as SEQ ID No. 2.

5. An isolated human BRCA2 truncated protein, wherein said truncated protein is selected from the group consisting of:

(A1) A polypeptide of amino acids 1720 to 1770 of SEQ ID NO. 2;

(A2) Polypeptide of 3160 th to 3418 th amino acid in SEQ ID NO. 2;

(A3) Polypeptide of 1700 th to 1800 th amino acid in SEQ ID NO. 2;

(A4) Polypeptide of 1700 th to 1838 th amino acid in SEQ ID NO. 2.

6. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of:

(a) A polynucleotide encoding the truncated protein of claim 5;

(b) A polynucleotide complementary to polynucleotide (a).

7. A vector comprising the polynucleotide of claim 6.

8. A genetically engineered host cell comprising the vector of claim 7 or having integrated into its genome the polynucleotide of claim 6.

9. A method for preparing BRCA2 truncated protein, comprising:

(a) Culturing the host cell of claim 8 under conditions suitable for expression;

(b) Isolating said BRCA2 truncated protein from the culture.

10. A kit, said kit comprising:

(b) A description of the detection method is given.